Enantiomers are asymmetric molecules that can exist in two different isomeric forms which have different configurations in space. Because they do not have a plane of symmetry, enantiomers are not identical with their mirror images; molecules which exist in two enantiomeric forms are chiral, which means that they can be regarded as occurring in “left” and “right” handed forms. The most common cause of chirality in organic molecules is the presence of a tetrahedral carbon bonded to four different substituents or groups. Such a carbon is referred to as a chiral center, or stereogenic center. A method for indicating the three-dimensional arrangement of atoms (or the configuration) at a stereogenic center is to refer to the arrangement of the priority of the groups when the lowest priority group is oriented away from a hypothetical observer: If the arrangement of the remaining three groups from the higher to the lower priority is clockwise, the stereogenic center has an “R” (or “D”) configuration; if the arrangement is counterclockwise, the stereogenic center has an “S” (or “L”) configuration.
Enantiomers have the same empirical chemical formula, and are generally chemically identical in their reactions. However, enantiomers show different chemical reactivity toward other asymmetric compounds, and respond differently toward asymmetric physical disturbances. The most common asymmetric disturbance is polarized light.
An enantiomer can rotate plane-polarized light; thus, the enantiomer is optically active. Two different enantiomers of the same compound will rotate plane-polarized light in the opposite direction; thus, the light can be rotated to the left or counterclockwise for a hypothetical observer (this is levarotatory or “1”, or minus or “−”) or it can be rotated to the right or clockwise (this is dextrorotatory or “d” or plus or “+”). The sign of optical rotation (+) or (−), is not related to the R,S designation) A mixture of equal amounts of two chiral enantiomers is called a racemic mixture, or racemate, and is denoted either by the symbol (+/−) or by the prefix “d,l” to indicate a mixture of dextrorotatory and levorotatory forms. Racemic mixtures show zero optical rotation because equal amounts of the (+) and (−) forms are present. But generally the presence of a single enantiomer rotates the light in only one direction; thus, a single enantiomer is referred to as optically pure.
Optically pure compounds are of interest as chiral synthons. One reason is that asymmetric molecules in living organisms are usually present in only one of their possible chiral forms. In contrast, when a chiral organic compound is synthesized in the laboratory, the synthetic reactions (in the absence of asymmetric catalysts) generally produce both chiral forms at an equal rate, leading to an equimolar, or racemic, mixture of the product isomers. The separation of a racemic mixture into its two constituent enantiomers is called resolution, but it is very difficult to separate a racemic mixture (one way is by reaction of a racemate with a standard asymmetric compound, and separating the resulting products (diastereomers) which have different physical properties, and then removing the standard asymmetric compound). However, the three-dimensional shape, or stereochemistry, of biomolecules is extremely important to their biological function. Moreover, enantiomers of the same structure may have very different biological effects. As an example, the drug thalidomide was synthesized and administered as a racemate; only one enantiomer was an effective anti-nausea drug, whereas the other enantiomer was an effective teratogen, which was tragically discovered after administration of the racemate to pregnant women.
Therefore, the synthesis of molecules for a biological function (such as a drug) preferably occurs from a single enantiomer which will result in the desired biologically active product. For example, chiral 1,2-propanediols are useful in the preparation of cardiovascular drugs, anti viral drugs, and enantiomerically pure crown ethers (Hoff et al. (1996) Tetrahedron: Asymmetry 7:3181-3186). These and related chiral compounds may also serve as synthons for chiral polymers, chromatography matrices, or as derivatization reagents for stereochemical analysis of chiral acids by LC or NMR. In addition to pharmaceutical and agricultural applications, optically active secondary alcohols, particularly those with asymmetric carbon containing fluoroalkyl groups (e.g., trifluormethyl-), are a material of interest in ferroelectric and anti-ferroelectric liquid crystals (U.S. Pat. No. 6,239,316 B1; EPO Application No. 99115154.9).
Optically pure compounds can be synthesized chemically, but asymmetric chemical synthesis often requires catalysts which are expensive and/or which may be environmentally deleterious; moreover, such syntheses are typically multi-step, and often limited to substrates containing the structural requirements for a given chemistry. Mixtures of isomers or enantiomers, resulting from typical chemical syntheses, can be resolved (or separated into pure enantiomers) either chemically or enzymatically. Chemical resolution of racemates has been described above. Enzymatic resolution of racemates relies upon a preference or selectivity of an enzyme for one isomer of a racemate as a substrate; the result is the formation of a product from predominantly the preferred substrate, while leaving the non-preferred substrate predominantly as the original isomer. The preference of an enzyme toward one isomer of a racemate is known as enantioselectivity. The use of esterases, lipases, and proteases to perform kinetic resolutions of mixtures of enantiomers is well known. However, enantioselective resolution of a mixture of enantiomers is highly dependent upon not only the choice of enzyme, but also upon the chemical structure of the enzyme substrates. The optimal choice of an enzyme to resolve a given substrate or a series of related substrates is therefore not easily predicted, but requires careful screening of a variety of enzymes while varying the chemical structure of potential substrates.
Some examples of enzyme catalyzed resolution of a few propylene glycol ethers and related derivatives have been described in the scientific and patent literature. A screening for enantioselective hydrolysis of difficult-to-resolve substrates, including the acetate and butyrate esters of (±)-1-methoxy-2-propanol, has been described recently (Baumann et al. (2000) Tetrahedron: Asymmetry 11:4781-4790). The authors reported that a lipase fraction B from Candida antarctica (CAL-B) catalyzed enantioselective hydrolysis of (R)-1-methoxy-2-propanol acetate yielding the corresponding (S)-acetate and (R)-alcohol of greater than 99% ee and in maximum yield (50%). Preparative scale reactions were apparently conducted at 50 ml scale with 5.28% (w/v) 1-methoxy-2-propanol acetate (calculated based on 20 mmol substrate, 50 mg enzyme at 1.0 mg/ml). Enantioselective acylation of 2.7% (w/v) (±)-1-methoxy-2-propanol in hexane with vinyl acetate as acyl donor was reported to give (R)-1-methoxy-2-propanol acetate in 98% ee and 20% yield substrate conversion (Baumann et al. (2000) Tetrahedron: Asymmetry 11:4781-4790).
Another example of an optical resolution using the CAL-B lipase (Novozyme 435) is the catalysis by enantioselective transesterification of 4-isopropyloxybutan-2-ol with vinyl propionate to yield (R)-(+)-4-isopropyloxybutane-2-propionate (U.S. Pat. No. 6,239,316 B1; EPO Application No.99115154.9). Chemical hydrolysis of the ester provided the corresponding (R)-alcohol in greater than 95% ee.
Individual propylene glycol ethers (for example, propylene glycol phenyl ether or 1-phenoxy-2-propanol) and their corresponding acetates (for example, propylene glycol phenyl ether acetate or 1-phenoxy-2-propyl acetate) have been the focus of biocatalytic resolution by a number of groups. Three Japanese patents have described the manufacture of both (R)- and (S)-1-aryloxy-2-alkanols by treatment of racemic acetates with whole cells, their preparations, or lipase from them (Japanese Patent Application Nos. JP 1991-262377, JP 1991-262378, and JP 1991-262379); much of the work described in the patents was later published (Yanase et al. (1993) Biosci. Biotech. Biochem. 57:1334-1337). In addition, hydrolytic resolution of the butanoic ester of 1-phenoxy-2-propanol has been studied with CAL-B and other lipases (Hoff et al. (1996) Tetrahedron: Asymmetry 7:3181-3186). Transesterification of 1-phenoxy-2-propanol with 2-chloroethylbutanoate and other acyl donors has been used in the development of a computer program for calculating enantiomer ratio and equilibrium constants in biocatalytic resolutions (Anthonsen et al. (1995) Tetrahedron: Asymmetry 6:3015-3022; Hoff et al. (1996) Tetrahedron: Asymmetry 7:3187-3192). The transesterification of 1-phenoxy-2-propanol (approx 3% w/v) has also been demonstrated with polymer activated forms of pig liver esterase (PLE) using vinyl propionate as acyl donor (Gais et al. (2001) J. Org. Chem. 66:3384-3396).
However, enantioselective resolution via enzymatic hydrolysis or acylation has not been reported for racemates of related propylene glycol alkyl (or aryl) ethers and their corresponding acetates; exemplary members of these related glycol ethers include but are not limited to propylene glycol ethyl ether, propylene glycol n-propyl ether, propylene glycol isopropyl ether, propylene glycol n-butyl ether, propylene glycol t-butyl ether, and their corresponding acetates. Nor have reaction conditions been described for either hydrolysis or acylation of propylene glycol methyl ether or propylene glycol phenyl ether or their corresponding acetates at high substrate concentrations which result in the formation of products in high enantiopurity and in high yields.
Glycol ethers and their corresponding acetates, and in particular, propylene glycol alkyl (or aryl) ethers and their corresponding acetates, have been particularly difficult to resolve in a commercially feasible manner. Therefore, it would be useful to develop processes for resolving racemic mixtures of propylene glycol alkyl (or aryl) ethers and their corresponding acetates, where the processes are enzymatic, result in a high degree of separation at a high yield, and are fast and economical.